LOCUS       sequence-486417-        7147 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Donor II vector in DASH and alpha1 vector in GoldenBraid/DASH, with 
            attPCC and attPTT-FRT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7147)
  AUTHORS   Zhao C, Stepanova AN, Alonso JM
  TITLE     DASH: a versatile and high-capacity gene stacking system for plant 
            synthetic biology.
  JOURNAL   Plant Biotechnol J. 2025 Jun 10. doi: 10.1111/pbi.70179.
  PUBMED    40493021
REFERENCE   2  (bases 1 to 7147)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Plant 
            Biotechnol J. 2025 Jun 10. doi: 10.1111/pbi.70179."
COMMENT     Sequence Label: pDASH-DII-alpha-1
FEATURES             Location/Qualifiers
     source          1..7147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..25
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     primer_bind     410..432
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     424..441
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     425..441
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    442..498
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(511..527)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(511..527)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(524..546)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    535..551
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(559..565,566..583,584..589))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    604..625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(753..786)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    793..817
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2118..2747
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3176..4249
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      4315..4509
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4745..4767)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4853..4993
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5008..5025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5179..5767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5259..5278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5855..6646,6647..6715))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQYFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6478..6497
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tggcaggata tattgtggtg taaacataac gaattcagta gtgccccaac tggggtaacc
       61 tccgagttct ctcagttggg ggcgtacgtc tcaggagaga gacccacagc ttgtctgtaa
      121 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
      181 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
      241 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg
      301 ctgcgcaact gttgggaagg gcgatcggcg caggcctctt cgctattacg ccagctggcg
      361 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga
      421 cgttgtaaaa cgacggccag tgaattcgag ctcggtaccc ggggatcctc tagagtcgac
      481 ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
      541 tccgctcaca attccacaca acatacgagc cggaagcata tagtgtaaag cctggggtgc
      601 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
      661 aaacctgtcg tgccagctgg ggtctctcgc tgtcatgaga cgagtagtgc cccaactggg
      721 gtaacctttg agttctctca gttgggggcg tagaagttcc tatactttct agagaatagg
      781 aacttcgaat tctgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta
      841 gaataatcgg atatttaaaa gggcgtgaaa aggtttatcc gttcgtccat ttgtatgtgc
      901 atgccaacca cagggttccc ctcgggatca aagtactttg atccaacccc tccgctgcta
      961 tagtgcagtc ggcttctgac gttcagtgca gccgtcatct gaaaacgaca tgtcgcacaa
     1021 gtcctaagtt acgcgacagg ctgccgccct gcccttttcc tggcgttttc ttgtcgcgtg
     1081 ttttagtcgc ataaagtaga atacttgcga ctagaaccgg agacattacg ccatgaacaa
     1141 gagcgccgcc gctggcctgc tgggctatgc ccgcgtcagc accgacgacc aggacttgac
     1201 caaccaacgg gccgaactgc acgcggccgg ctgcaccaag ctgttttccg agaagatcac
     1261 cggcaccagg cgcgaccgcc cggagctggc caggatgctt gaccacctac gccctggcga
     1321 cgttgtgaca gtgaccaggc tagaccgcct ggcccgcagc acccgcgacc tactggacat
     1381 tgccgagcgc atccaggagg ccggcgcggg cctgcgtagc ctggcagagc cgtgggccga
     1441 caccaccacg ccggccggcc gcatggtgtt gaccgtgttc gccggcattg ccgagttcga
     1501 gcgttcccta atcatcgacc gcacccggag cgggcgcgag gccgccaagg cccgaggcgt
     1561 gaagtttggc ccccgcccta ccctcacccc ggcacagatc gcgcacgccc gcgagctgat
     1621 cgaccaggaa ggccgcaccg tgaaagaggc ggctgcactg cttggcgtgc atcgctcgac
     1681 cctgtaccgc gcacttgagc gcagcgagga agtgacgccc accgaggcca ggcggcgcgg
     1741 tgccttccgt gaggacgcat tgaccgaggc cgacgccctg gcggccgccg agaatgaacg
     1801 ccaagaggaa caagcatgaa accgcaccag gacggccagg acgaaccgtt tttcattacc
     1861 gaagagatcg aggcggagat gatcgcggcc gggtacgtgt tcgagccgcc cgcgcacctc
     1921 tcaaccgtgc ggctgcatga aatcctggcc ggtttgtctg atgccaagct ggcggcctgg
     1981 ccggccagct tggccgctga agaaaccgag cgccgccgtc taaaaaggtg atgtgtattt
     2041 gagtaaaaca gcttgcgtca tgcggtcgct gcgtatatga tgcgatgagt aaataaacaa
     2101 atacgcaagg ggaacgcatg aaggttatcg ctgtacttaa ccagaaaggc gggtcaggca
     2161 agacgaccat cgcaacccat ctagcccgcg ccctgcaact cgccggggcc gatgttctgt
     2221 tagtcgattc cgatccccag ggcagtgccc gcgattgggc ggccgtgcgg gaagatcaac
     2281 cgctaaccgt tgtcggcatc gaccgcccga cgattgaccg cgacgtgaag gccatcggcc
     2341 ggcgcgactt cgtagtgatc gacggagcgc cccaggcggc ggacttggct gtgtccgcga
     2401 tcaaggcagc cgacttcgtg ctgattccgg tgcagccaag cccttacgac atatgggcca
     2461 ccgccgacct ggtggagctg gttaagcagc gcattgaggt cacggatgga aggctacaag
     2521 cggcctttgt cgtgtcgcgg gcgatcaaag gcacgcgcat cggcggtgag gttgccgagg
     2581 cgctggccgg gtacgagctg cccattcttg agtcccgtat cacgcagcgc gtgagctacc
     2641 caggcactgc cgccgccggc acaaccgttc ttgaatcaga acccgagggc gacgctgccc
     2701 gcgaggtcca ggcgctggcc gctgaaatta aatcaaaact catttgagtt aatgaggtaa
     2761 agagaaaatg agcaaaagca caaacacgct aagtgccggc cgtccgagcg cacgcagcag
     2821 caaggctgca acgttggcca gcctggcaga cacgccagcc atgaagcggg tcaactttca
     2881 gttgccggcg gaggatcaca ccaagctgaa gatgtacgcg gtacgccaag gcaagaccat
     2941 taccgagctg ctatctgaat acatcgcgca gctaccagag taaatgagca aatgaataaa
     3001 tgagtagatg aattttagcg gctaaaggag gcggcatgga aaatcaagaa caaccaggca
     3061 ccgacgccgt ggaatgcccc atgtgtggag gaacgggcgg ttggccaggc gtaagcggct
     3121 gggttgcctg ccggccctgc aatggcactg gaacccccaa gcccgaggaa tcggcgtgag
     3181 cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga
     3241 gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg
     3301 tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc
     3361 cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc
     3421 gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg
     3481 tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca
     3541 cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact
     3601 gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa
     3661 gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga
     3721 tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt
     3781 tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga
     3841 agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga
     3901 gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct
     3961 gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct
     4021 ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg
     4081 cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc
     4141 aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt
     4201 catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca
     4261 gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggactct ttcctgtgga
     4321 tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa
     4381 cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa
     4441 aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc
     4501 ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg
     4561 gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc
     4621 aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc
     4681 actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg
     4741 aaaacctctg acacatgcag ctcccggtga cggtcacagc ttgtctgtaa gcggatgccg
     4801 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     4861 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     4921 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     4981 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5041 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5101 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5161 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5221 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5281 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5341 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5401 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5461 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5521 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5581 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5641 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5701 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5761 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5821 acgttaaggg attttggtca tgcattctag gtgattacca atgcttaatc agtgaggcac
     5881 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5941 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgggacc
     6001 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6061 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6121 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6181 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6241 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6301 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6361 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6421 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6481 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6541 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6601 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6661 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atattgaata ctcataacag
     6721 aacttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat
     6781 aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca
     6841 gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg
     6901 aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc
     6961 cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg
     7021 gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg
     7081 atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc
     7141 tggctgg
//
